Effects of experimental calcium availability, egg parameters and laying order on Great Tit Parus major eggshell pigmentation patterns
Abstract
Many bird species lay eggs speckled with protoporphyrin-based spots, however, for most of them the function of eggshell spotting is unknown. A plausible hypothesis is that protoporphyrin might have a structural function in strengthening the eggshell and is therefore deposited when calcium is scarce. In this study, we experimentally provided Great Tit Parus major females with supplemental calcium to examine its effect on the protoporphyrin-based maculation of their eggs. In addition, we studied variation in eggshell pigmentation patterns in relation to other egg parameters and laying order. Calcium-supplemented females laid larger eggs but shell thickness was not significantly affected by the treatment. Calcium supplementation may reduce the time and energy females devote to searching for calcium-rich material, so that they can collect more nutrients and so lay larger eggs. Furthermore, pigment darkness was associated with egg volume and shape, which suggests that female quality and environmental food availability may also influence the shell pigmentation pattern. Within clutches, later-laid eggs had larger and darker spots that were distributed more unevenly on the shell surface. This within-clutch pattern could be explained by the increase in egg volume and egg shape and a decline in shell thickness with egg-laying order, which characteristics were all related to shell-spotting pattern. Eggs with a coronal ring had thinner shells, but pigment intensity and spot size were not related to shell thickness. Thus, our results suggest that concentrated spotting distribution may have a mechanical function, supporting the structural-function hypothesis.
Variation in avian eggshell coloration has traditionally been interpreted as a response to selection pressures imposed by nest predators and brood parasites, although other functions have also been proposed (Underwood & Sealy 2002, Reynolds et al. 2009). Understanding the source of this variation is crucial for understanding the functions of eggshell patterning. Although much evidence supports a genetic basis for eggshell pigmentation in birds (Collins 1993, Gosler et al. 2000), environmental factors, such as food and calcium availability, or the condition of the laying female may also influence egg coloration (Gosler et al. 2005, Moreno et al. 2006, Avilés et al. 2007, Martínez-de la Puente et al. 2007). Egg coloration may also vary within clutches with the order of laying, due to either depletion of pigments or other environmental or physiological factors (Lowther 1988, Moreno et al. 2005, López de Hierro & De Neve 2010, Martínez-Padilla et al. 2010).
Many bird species lay white eggs speckled with red or brown spots. For most species, the function of the pigmentation is unknown. Protoporphyrin is responsible for the red and brown egg colours (Kennedy & Vevers 1976, Mikšík et al. 1996, Gorchein et al. 2009). It is a metabolite intermediate in the biosynthesis of haem (Baird et al. 1975) or may derive from the transformation of haem (Wang et al. 2009). Eggshell protoporphyrin may be produced by red blood cells (Kennedy & Vevers 1973) and later accumulate in the shell gland before oviposition (Soh & Koga 1994, Wang et al. 2009), or it may be synthesized in the epithelial cells of the shell gland (Baird et al. 1975, Zhao et al. 2006, Wang et al. 2007). Protoporphyrin occurs at a range of depths within the eggshell (Jagannath et al. 2008, Bulla et al. 2012).
Tits (Paridae) are secondary hole-nesting birds, so the protoporphyrin-based spottiness of their eggs is unlikely to function as an adaptation to conceal eggs from nest predators. Moreover, there is apparently no intraspecific brood parasitism in these species (Kempenaers et al. 1995, Griffith et al. 2009), and females do not detect and eject different-looking eggs (R. Hargitai, pers. obs.). Nevertheless, we cannot rule out that the development of spotting was favoured by avoidance of nest predators or brood parasitism in the past. However, the question arises why tits have maintained eggshell spotting as cavity nesters.
One possible function of protoporphyrin-based eggshell spotting is to strengthen the shell (Solomon 1997, Gosler et al. 2005). Shell strength is primarily affected by shell thickness, which may reflect calcium availability, a critical source for small passerines at the time of egg formation (Graveland & van Gijzen 1994, Bureš & Weidinger 2003). Solomon (1997) suggested a structural reinforcement role for protoporphyrin in the eggshell because of its structural similarity to phthalocyanines, lubricants in solid-state engineering. According to this hypothesis, protoporphyrin between the calcite crystals could act as a ‘cushion’, making the eggshell more resistant. Studies of Great Tits Parus major in a British population nesting across a soil calcium gradient revealed that the darkness of protoporphyrin speckling on the birds' eggs correlated negatively with shell thickness, which varied with calcium availability (Gosler et al. 2005). With declining calcium, shell thickness also became less uniform, and protoporphyrin occurred precisely where the shell was thinner and in proportion to the degree of shell thinning (Gosler et al. 2005). That study supported the hypotheses that protoporphyrin might have a function as an intercrystalline shock absorber, and share a protein carrier with calcium within the shell gland, so being deposited specifically when calcium is scarce (Solomon 1997).
However, support for the structural–function hypothesis is still ambiguous, as different studies showed contrasting results both among and within species. For instance, Sanz and García-Navas (2009) reported that the darkness of the eggshell patterning positively correlated with shell thickness in the Blue Tit Cyanistes caeruleus. In another study of Blue Tits, experimental calcium supplementation did not affect pigment darkness, but the distribution of spots was more homogeneous in the eggs of the supplemented group (García-Navas et al. 2010). Similarly, shell spot darkness and shell thickness showed no relationship in the Northern Lapwing Vanellus vanellus (Bulla et al. 2012) and in an Estonian population of Great Tits (Mägi et al. 2012). Moreover, in the Mexican Jay Aphelocoma ultramarina (Berg et al. 2009) and in an Estonian population of Great Tits (Mägi et al. 2012), local soil calcium level and pattern of egg speckling showed no significant relationship. Adaptation to calcium shortage, including the deposition of protoporphyrin pigments to the eggshell, may vary between different bird species or geographical populations of the same species owing to possible differences in shell structure, environmental circumstances and evolutionary histories (Tilgar et al. 2002, Reynolds et al. 2004). Research assessing the correlation between eggshell maculation pattern and thickness across a range of bird populations is needed to derive conclusions on the generality or specificity of the structural–function hypothesis (Solomon 1997, Gosler et al. 2005).
In this study, we experimentally provided Great Tits with supplemental calcium to examine its effect on the protoporphyrin-based maculation of their eggs in order to assess whether shell pigmentation is related to the female's calcium availability. We also analysed the relationship between pigmentation pattern and egg parameters, including volume, shape and shell thickness. Apart from studying spot size, spot intensity and spotting distribution, we included a variable for the presence or absence of concentrated spotting at the blunt end of the egg (coronal ring) in the analyses, which, to our knowledge, has not previously been assessed in the framework of the structural–function hypothesis (Solomon 1997, Gosler et al. 2005). Based on the above hypothesis, we predicted that calcium-supplemented Great Tit females would lay thicker-shelled eggs with smaller and paler spots and without a coronal ring compared with control birds. Moreover, we expected to find an association between shell thickness and pigmentation pattern. In addition, our aim was to study variation in egg characteristics in relation to laying order. We expected that the within-clutch pigmentation pattern would change according to the increase or decline of eggshell thickness across the laying sequence.
Methods
General field procedures
The study was carried out on a nestbox-breeding population of Great Tits in a woodland dominated by Sessile Oak Quercus petraea in the Pilis Mountains (47°43′N, 19°01′E), Hungary, in 2009 and 2011. The woodland has clayey brown forest soil on volcanic stones (pH 5–6), suggesting that the availability of calcium in the area is low (< 1000 mg per 100 g soil; Higham & Gosler 2006). Eggs were numbered with a waterproof marker according to laying order and four (the 1st, 4th, 7th and 10th eggs) were collected before incubation began and replaced with dummy eggs. In 2009, the 10th eggs could not be collected in three nests as females had started incubation, and we did not wish to risk nest desertion by disturbing them. Egg length and width were measured with a calliper (to the nearest 0.1 mm), volume was estimated using Hoyt's formula (Hoyt 1979, volume = length × width2 × 0.51). Shape was calculated as egg width/length (Gosler et al. 2005).
Estimation of natural calcium availability: snail study
We estimated natural calcium availability and possible calcium limitation in the study area by assessing the abundance of land snails, as it has been reported that soil calcium concentration is related to their abundance and diversity (Graveland et al. 1994, Jubb et al. 2006). We collected 20 quadrats of 50 × 50 cm from the litter layer and the top 1–2 cm of the mineral soil following Mänd et al. (2000) and García-Navas et al. (2010). Samples were collected in the neighbourhood of the study nestboxes in dry weather in mid-April 2011. Samples were searched by eye in the laboratory for 25–35 min, and snail shells found in the material were counted and measured.
Calcium-supplementation experiment
Females were supplemented with calcium by attaching a small feeding cup outside the nestbox on the day when the first egg was laid. In 2009, the cups were filled with c. 1 g of limestone granules and small pieces of white snail shell (supplemented group) or were left empty (control group). In 2011, supplemented birds were provided with cuttlefish bone (also attached to the top of the nestbox) and small pieces of white domestic hen eggshells (1–2 g) in a feeding cup. Feeders were checked every day in 2011 and every 1 or 2 days in 2009, and if necessary were refilled so that birds were provided ad libitum with calcium-rich items. We recorded the small pecks on the provided cuttlefish bone in 2011: in all supplemented nests we found small pecks on average 3.2 (±1.6) days after the start of the experiment, suggesting that birds fed on the supplemented material. Birds were fed until clutch completion, and the cups and cuttlefish bones were removed at the beginning of incubation. Consumption of experimentally provided supplemental calcium has been observed in several passerine species including the Great Tit (e.g. Graveland & Drent 1997, Tilgar et al. 2002). We assume that male Great Tits did not consume the supplemented calcium-rich material because their calcium requirements are much lower than those of egg-laying females (Graveland & Berends 1997). Supplemented and control nestboxes were at least 100 m apart (Graveland & Drent 1997). The experiment was started with 25 nests (15 supplemented and 10 control) in 2009. However, five nests were deserted during the egg-laying period, which resulted in 12 supplemented and eight control nests in 2009. In 2011, the experiment was repeated with 14 supplemented and 11 control nests.
Eggshell pigmentation scoring
Two photographs were taken of the eggs (side and bottom views) with an HP Photosmart M407 digital camera in the field in 2009, and in the laboratory in 2011. Eggshells were scored from photographs by one observer (R.H.) blind to the experimental treatment of the clutches (Gosler et al. 2005). Eggshell pigmentation was scored in three categories from the side view of the eggs: pigment intensity (I: from 1 for the palest to 5 for the darkest), average spot size (S: scored in 0.5 increments from 1 for the smallest spots to 3 for the largest spots), and spotting distribution (D: from 1 for the aggregated spotting distribution to 5 for even spotting distribution). The within-clutch repeatability values of pigmentation variables were significant (I: F43,272 = 8.15, P < 0.001, r = 0.50; S: F43,272 = 5.38, P < 0.001, r = 0.38; D: F43,272 = 3.66, P < 0.001, r = 0.27). The presence or absence of concentrated spotting at the blunt end of the egg (coronal ring) was also registered (0: no coronal ring, 1: coronal ring) on the basis of the side and bottom views of the egg.
We initially performed a principal component analysis (PCA) with the three score variables (I, S, D). The first principal component (PC1; eigenvalue: 1.92) explained 64% of the total variation, PC2 explained 21% of the total variation (eigenvalue: 0.64), and PC3 explained a further 15% of the total variation (eigenvalue: 0.44). However, the factor loadings for the three original variables were similar in PC1 (I: 0.83, S: 0.83, D: –0.74), so we cannot attribute PC1 to ‘darkness’ or ‘spread’ sensu earlier studies (Gosler et al. 2005, Sanz & García-Navas 2009, García-Navas et al. 2010, López de Hierro & De Neve 2010, Mägi et al. 2012). Furthermore, it is generally proposed that only factors with eigenvalues greater than 1 should be retained after PCA (Kaiser criterion; Kaiser 1960). Therefore, we decided to analyse the three original score variables and the presence of a coronal ring separately.
Eggshell thickness measurement
Pieces of eggshell were taken from the equatorial region of four eggs per clutch. Shell thickness was measured with a Moore & Wright digital micrometer (MW255-01DDL, Bowers Metrology Ltd., Bradford, UK) to a precision of 0.001 mm at three spotted and three unspotted points on the shell. Eggshell thickness measurements made using a micrometer have been shown to be as reliable as those obtained using scanning electron microscopy (Igic et al. 2010). Variation in shell thickness was greater among than within eggs (2009: F79, 880 = 22.77, P < 0.001, r = 0.65; 2011: F99, 1100 = 40.35, P < 0.001, r = 0.77), indicating that these measurements were repeatable (Lessells & Boag 1987). We used the mean of the six measurements as the average shell thickness of an egg.
Statistical analyses
The effect of calcium-supplementation on egg parameters was tested on the 10th-laid egg with year and treatment as fixed factors and a year * treatment interaction in a general linear model. We also assessed whether there was a difference in the characteristics of first-laid eggs between the supplemented and control groups, where we expect none. The effect of calcium-supplementation on the presence of a coronal ring was tested with a generalized linear model with a binomial distribution and logit-link function. Associations of eggshell pigmentation pattern (I, S, D) and presence of a coronal ring with egg parameters were analysed with general linear mixed models, and a generalized linear mixed model with binomial distribution and logit link function, respectively. Nest identity was fitted as a random factor, year as a fixed factor, and egg volume, shape, shell thickness and laying order as fixed covariates. Interactions between year and covariates were also tested, but only significant interactions are reported. Eggshell thickness differed significantly between years (see later), so we standardized this variable within years before analysing it as a covariate. One clutch contained eggs without spots in 2009, and we omitted this clutch from analyses. The effect of laying order on egg volume, shape and shell thickness was analysed with general linear mixed models including year, laying order and a year * laying order interaction, and nest identity as a random factor. We applied maximum likelihood estimation, which is considered more suitable for mixed models than restricted maximum likelihood estimation (Singer & Willett 2003). In all models, a stepwise analysis based on a backward deletion procedure was employed, removing non-significant (P > 0.05) effects from the model one by one in decreasing order of P. To avoid non-significance due to overparameterization, we re-entered non-significant effects to the final model one by one, and present these F- and P-values. Analyses were performed in spss 17.0 (SPSS Inc., Chicago, IL, USA) and sas 9.1 (SAS Inc.).
Results
Calcium supplementation
Land snails were almost completely absent in the litter samples. In total, two snail shells (diameter: 0.5 cm) were found in the 20 quadrat samples.
We found no difference in shell pigmentation pattern, shell thickness, egg shape and egg volume of first-laid eggs between calcium-supplemented and control groups (all P > 0.10). The 10th-laid eggs of clutches of the calcium-supplemented group, where we expected the largest effect of the supplementation, were larger, but unexpectedly tended to have thinner shells than those of the control group (Table 1). There was a significant year * treatment interaction on pigment intensity (Table 1): calcium-supplemented birds laid eggs with darker spots in 2011 (F1,25 = 8.01, P = 0.009) but no significant relationship was detected in 2009 (F1,19 = 0.93, P = 0.35). Calcium supplementation treatment did not affect eggshell spot size, spotting distribution, presence of a coronal ring, egg shape (Table 1) or clutch size (F1,42 = 0.15, P = 0.70).
| Ca-supplemented | Control | F | df | P | |
|---|---|---|---|---|---|
| Pigment intensity | 3.48 ± 0.20 | 3.27 ± 0.25 | |||
| Treatment | 0.44 | 1,44 | 0.51 | ||
| Year | 0.19 | 1,44 | 0.66 | ||
| Treatment * Year | 5.77 | 1,44 | 0.021 | ||
| Spot size | 2.15 ± 0.08 | 2.17 ± 0.09 | |||
| Treatment | 0.01 | 1,44 | 0.91 | ||
| Year | 2.78 | 1,44 | 0.10 | ||
| Treatment * Year | 0.38 | 1,44 | 0.54 | ||
| Spotting distribution | 3.04 ± 0.24 | 2.78 ± 0.29 | |||
| Treatment | 0.47 | 1,44 | 0.50 | ||
| Year | 1.12 | 1,44 | 0.30 | ||
| Treatment * Year | 2.57 | 1,44 | 0.12 | ||
| Presence of a coronal ring | 0.31 ± 0.09 | 0.39 ± 0.12 | |||
| Treatment | 0.31 (Wald χ2) | 1 | 0.58 | ||
| Year | 0.12 (Wald χ2) | 1 | 0.74 | ||
| Treatment * Year | 0.001 (Wald χ2) | 1 | 0.97 | ||
| Egg volume (cm3) | 1.61 ± 0.02 | 1.54 ± 0.03 | |||
| Treatment | 4.20 | 1,44 | 0.046 | ||
| Year | 0.24 | 1,44 | 0.63 | ||
| Treatment * Year | 0.49 | 1,44 | 0.49 | ||
| Egg shape (width/length) | 0.76 ± 0.006 | 0.75 ± 0.007 | |||
| Treatment | 2.49 | 1,44 | 0.12 | ||
| Year | 4.17 | 1,44 | 0.047 | ||
| Treatment * Year | 0.28 | 1,44 | 0.60 | ||
| Eggshell thickness (μm) | 78.18 ± 0.66 | 80.13 ± 0.79 | |||
| Treatment | 3.66 | 1,41 | 0.063 | ||
| Year | 4.31 | 1,41 | 0.044 | ||
| Treatment * Year | 0.03 | 1,41 | 0.88 |
- Values are given as means ± 1 sd. Significant differences are indicated in bold.
Eggshell pigmentation pattern and egg parameters
The presence of a coronal ring showed a significant relationship with shell thickness: eggs with a coronal ring had thinner shells than those without a coronal ring (Table 2). In addition, the coronal ring was more frequent in the eggs in 2011 than in 2009 (presence of coronal ring: 2009: 56%, 2011: 76%; Table 2), when average shell thickness was lower (general linear mixed model: F1,44.04 = 19.66, P < 0.001; 2009: 81.5 ± 0.6 μm; 2011: 77.7 ± 0.5 μm). Spotting distribution also tended to be related to eggshell thickness, indicating that thinner-shelled eggs showed a more aggregated spotting distribution (Table 2). However, pigment intensity and spot size were not related to eggshell thickness (Table 2). There was no difference in the thickness of pigmented and unpigmented shell areas (paired t-test: t = −0.21, df = 176, P = 0.83). Furthermore, larger and more spherical eggs contained darker pigment spots on their shell (Table 2). Larger eggs tended to have a coronal ring, but egg shape was not related to presence of a coronal ring (Table 2).
| Dependent variable | Independent variables | df | F | P (estimate) |
|---|---|---|---|---|
| Pigment intensity | Egg volume | 1,166.09 | 8.77 | 0.004 (2.11) |
| Egg shape | 1,170.97 | 9.76 | 0.002 (9.34) | |
| Eggshell thickness | 1,159.72 | 0.04 | 0.84 | |
| Laying order | 1,137.33 | 23.91 | < 0.001 (0.08) | |
| Year | 1,44.00 | 0.01 | 0.93 | |
| Spot size | Egg volume | 1,164.99 | 1.48 | 0.23 |
| Egg shape | 1,140.55 | 2.53 | 0.11 | |
| Eggshell thickness | 1,171.00 | 0.32 | 0.57 | |
| Laying order | 1,128.50 | 75.66 | < 0.001 (0.06) | |
| Year | 1,44.26 | 0.76 | 0.39 | |
| Spotting distribution | Egg volume | 1,138.16 | 1.72 | 0.19 |
| Egg shape | 1,110.52 | 0.09 | 0.77 | |
| Eggshell thickness | 1,163.45 | 2.93 | 0.089 (0.15) | |
| Laying order | 1,130.98 | 22.71 | < 0.001 (−0.10) | |
| Year | 1,44.54 | 0.78 | 0.38 | |
| Presence of a coronal ring | Egg volume | 1,126 | 2.96 | 0.088 (4.02) |
| Egg shape | 1,126 | 0.12 | 0.73 | |
| Eggshell thickness | 1,128 | 11.61 | < 0.001 (−0.76) | |
| Laying order | 1,127 | 3.78 | 0.054 (0.10) | |
| Year | 1,128 | 4.92 | 0.028 |
- No year * independent variable interactions were significant. Significant effects are indicated in bold. Estimates of the significant fixed effects indicate the direction of the relationship between the dependent variable and the covariate.
Within-clutch variation
Later-laid eggs had larger and darker spots distributed more unevenly on the shell surface than earlier-laid eggs within a clutch (Table 2, Fig. 1). In addition, within clutches, later-laid eggs were more likely to have a coronal ring (Table 2). Egg volume and shape index increased whereas shell thickness declined with laying order: later-laid eggs were larger, more spherical and had thinner shells (egg volume: F1,298.31 = 63.49, P < 0.001, estimate: 0.009; egg shape: F1,299.64 = 11.97, P = 0.001, estimate: 0.009; eggshell thickness: F1,133.71 = 6.65, P = 0.011, estimate: −0.16).
Discussion
Calcium supplementation experiment
During egg-laying, female birds require large amounts of calcium-rich material, as their normal food usually does not contain sufficient calcium for shell formation (Graveland & van Gijzen 1994, Graveland & Berends 1997, Bureš & Weidinger 2003). Female passerines do not store metabolizable calcium in their skeleton and must obtain it daily during the laying period (Graveland & van Gijzen 1994). A shortage of calcium during egg formation could result in thinner-shelled eggs, and thus poor hatchability due to excessive water loss or shell breakage (Graveland et al. 1994, Eeva & Lehikoinen 1995, Higham & Gosler 2006). Our estimation of local land snail availability suggests a possible calcium limitation in our study area. However, calcium-supplemented Great Tit females did not lay eggs with thicker shells. Although Great Tits mainly use snail shells as a source of calcium for eggshell formation (Graveland et al. 1994), it is possible that in the case of land snail shortage they consume more calcium-rich arthropods such as millipedes and woodlice (Graveland & van Gijzen 1994, Bureš & Weidinger 2003) or use alternatives such as bones, teeth and anthropogenic calcium sources (Payne 1972, Graveland 1996). This foraging flexibility of Great Tits could explain why, as in other studies of Great Tits (Tilgar et al. 1999, Mänd et al. 2000, Mägi et al. 2012), experimental manipulation of calcium availability resulted in no increase in shell thickness in spite of apparently low soil calcium content in the area.
However, acquiring sufficient calcium is time-consuming even in a calcium-rich environment, so there may be a trade-off between time spent searching for calcium and for other nutrients (lipids, proteins) (Graveland & Berends 1997). We found that calcium-supplemented Great Tit females laid larger eggs. Because calcium supplementation may reduce the time and energy females devote to seeking calcium-rich material, they may have more time and energy for collecting other nutrients, and thereby lay larger eggs. Previous experimental studies also found that calcium-supplemented birds laid larger eggs (Tilgar et al. 1999, Mänd & Tilgar 2003). Furthermore, Graveland and van Gijzen (1994) showed that Great Tit eggs in a calcium-rich habitat were larger. The slightly thinner shells of the eggs laid by calcium-supplemented females in our study are unexpected and difficult to explain, but may be the result of the higher increase in egg volume and thus amount of calcium required for the shell than the amount of calcium consumed by supplemented females.
Calcium-supplemented birds laid eggs with darker spots in 2011, but not in 2009. Mägi et al. (2012) also found that calcium-supplemented Great Tit females laid darker pigmented eggs in an Estonian population in one study year, but not another. The partial positive effect of calcium supplementation on shell pigment intensity is in contrast to the main prediction of the structural-function hypothesis (Gosler et al. 2005). However, shell thickness was not affected by treatment in the Estonian Great Tit population (Mägi et al. 2012), whereas in our Hungarian population, calcium-supplemented Great Tits tended to lay slightly thinner-shelled eggs than control birds. Therefore, it cannot be concluded that the darker shell pigmentation of supplemented birds was associated with higher calcium deposition in the shell. Therefore, we suggest that experimentally elevated calcium availability may have influenced shell pigmentation pattern indirectly, probably through increasing egg volume, which was positively related to shell pigment intensity in our study.
In the Blue Tit, no significant difference was found in eggshell pigmentation pattern between calcium-supplemented and control groups when entire clutches were analysed, although shell thickness was significantly higher in the supplemented group (García-Navas et al. 2010). However, when only the eggs laid after calcium intake were considered, those in calcium-supplemented clutches showed a more homogeneous spotting distribution than those in control nests (García-Navas et al. 2010). This matches our finding that shell thickness is related to eggshell spotting distribution (see below).
Eggshell pigmentation pattern and egg parameters
We found that thinner-shelled Great Tit eggs were characterized by concentrated spotting at the blunt end of the egg (coronal ring), and also tended to show an uneven overall spotting distribution. In summary, our results showed that the coronal ring in Great Tit eggs was more frequent in a year when average shell thickness was lower. Likewise, Gosler et al. (2005) reported that shell pigment spread correlated with shell thickness in a British Great Tit population: pigment spots were more concentrated in eggs with a thinner-shelled shoulder region. In accordance with these results, eggs with thicker shells also had a more homogeneous spotting distribution in the Blue Tit (Sanz & García-Navas 2009, García-Navas et al. 2010).
A coronal ring can be found in the eggs of several small passerine species (Newton 1896, Lack 1968) and it has been suggested to have a mechanical function (Solomon 1997, Gosler et al. 2005, 2011). As hatching occurs at the shoulder region, it may be an adaptive trait that this area is thinner than other areas of the egg (Gosler et al. 2005). If protoporphyrin has a structural function in reinforcing the eggshell, more pigment spots are expected to be concentrated on that area of thinner-shelled eggs. Our result is consistent with earlier studies of tit species (Gosler et al. 2005, Sanz & García-Navas 2009, García-Navas et al. 2010, but see Mägi et al. 2012), suggesting that occurrence of spotting concentrated at the blunt end of the egg has a structural function.
Contrary to Gosler et al. (2005), the thickness of pigmented and unpigmented shell areas was not significantly different in our study, so our work does not suggest that protoporphyrin pigment is deposited exactly on those parts of the shell where calcium deposition is less intense. However, we should note that the measured unpigmented and pigmented areas were not adjacent to each other in our study. Also, as internalized spots may be found at thinner shell areas than superficial spots (Bulla et al. 2012), a more subtle classification of spots may yield different results. In contrast to our results, in the Eurasian Sparrowhawk Accipiter nisus (Jagannath et al. 2008), the Black-headed Gull Chroicocephalus ridibundus (Maurer et al. 2011) and the Northern Lapwing (Bulla et al. 2012), the eggshell was thinner at a spot than at a neighbouring unpigmented area.
We observed that larger and more spherical eggs showed darker shell pigmentation. In agreement with these results, Gosler et al. (2005) also found that larger Great Tit eggs had darker pigmentation, although egg shape was not related to pigment intensity in that study. Larger and more spherical eggs may contain more nutrients, and if nutrient availability positively affects protoporphyrin pigment production, we may expect a positive association between egg volume, egg shape and shell pigment darkness. In agreement with this hypothesis, in the Common Kestrel Falco tinnunculus, egg size was positively related to degree of shell pigmentation in food-supplemented pairs, but not in control pairs, suggesting that protoporphyrin pigment deposition is costly for Kestrels and is related to nutrient availability (Martínez-Padilla et al. 2010). Alternatively, as eggshell pigment darkness has a high genetic component in the Great Tit (Gosler et al. 2000), it is possible that better quality females are able to lay darker-pigmented and also larger eggs. In this case, darker pigmentation may reflect female quality rather than environmental circumstances. In contrast to these findings, heavier eggs tended to have paler spots in the Blue Tit (Martínez-de la Puente et al. 2007), suggesting that a lower level of protoporphyrin was deposited into heavier and probably larger eggs.
Within-clutch variation
Later-laid eggs had larger and darker spots distributed more unevenly on the shell surface within a clutch. Gosler et al. (2005) also found that pigment darkness increased with egg-laying sequence in a British Great Tit population, although in the House Sparrow Passer domesticus and the Common Kestrel, opposite within-clutch tendencies were found (López de Hierro & De Neve 2010, Martínez-Padilla et al. 2010). According to Gosler et al. (2005), the increase in shell darkness with egg-laying sequence could be explained as a compensation for the thinning of the shell with the order of eggs. In our Great Tit population, later-laid eggs were larger and more spherical and their shells were thinner. This within-clutch pattern of egg parameters could explain the increased pigment intensity and more concentrated spotting distribution of later-laid eggs within a clutch, because egg volume and shape correlated with pigment intensity, whereas shell thickness was related to spotting distribution. As information on the within-clutch variation of protoporphyrin-based spotting pattern is relatively scarce, more studies are needed.
Our results showed that the presence of a coronal ring was related to shell thickness, whereas pigment darkness was associated with egg volume and shape. Thus, in agreement with earlier studies of tit species (Gosler et al. 2005, Sanz & García-Navas 2009, García-Navas et al. 2010, but see Mägi et al. 2012), our work suggests that aggregated spotting distribution on the shell is a functional adaptation related to thin shells. The association between egg volume, egg shape and shell pigmentation darkness suggests that female quality and environmental food availability may also influence shell pigmentation pattern, which could be tested experimentally in future. Studies on a broad taxonomic range of bird species and populations breeding in different conditions of calcium and food availability would provide information on the generality of these findings on protoporphyrin-based eggshell spotting.
We are grateful to Gy. Blázi, R. Főző, L.Z. Garamszegi, G. Hegyi, D. Kiss, M. Laczi, G. Markó, B. Rosivall, E. Szász and E. Szöllősi for help with fieldwork. We thank two anonymous reviewers for their helpful comments. The Middle-Danube-Valley Inspectorate for Environmental Protection, Nature Conservation and Water Management kindly gave permission for this study (KTVF 23056-2/2009 and KTVF 22739-4/2011). This study was supported by the Hungarian Scientific Research Fund (OTKA, grants no. K75618 and PD100304), the Bolyai János Research Fellowship to R.H., the Erdők a Közjóért Alapítvány and the Pilis Park Forestry.
